IMCD cells transport protons, mediated by an H-ATPase, and H2O mediated by aquaporin-2 (AQP2) across their apical membrane. In our cultured line of IMCD cells, both of these processes are controlled by regulated exocytic insertion and endocytic retrieval of vesicles that carry either an H-ATPase or AQP2 as cargo in their membranes, but not both. It is probable that SNARE proteins, in part, mediate the targeting and fusion of these vesicles to the apical membrane. However, despite the similarity of the postulated targeting-fusion system, exocytosis of H-ATPase and AQP2 are independently regulated. The question then is how does a polar renal epithelial cell target two distinct cargo-laden vesicles to the apical membrane utilizing similar docking-fusion proteins? To answer this question, we propose the following hypotheses which will be the focus of the current proposal: 1) the minimal machinery, the SNAREpin, required for targeting and fusion of H-ATPase or AQP2 vesicle subtypes to the apical membrane consists of a distinct set of v & tSNAREs; 2) the cargo proteins (H+-ATPase and AQP2), per se, participate in the regulation, targeting and exocytic insertion of their carrier vesicles and 3) regulated exocytosis of these vesicles is not only initiated but proceeds by different signal cascades that modify (phosphorylate) dissimilar proteins in either the vesicles or target (apical) membrane. These hypotheses will be evaluated in our IMCD cell line, an excellent model system for the study of terminal medullary collecting duct function. These cells in culture express many if not all of the acid-base transporters that regulate H+ secretion and vasoressin regulated H20 permeability regulated by apically targeted AQP2. Thus these studies provide a unique opportunity for determining the cellular processes that leads to the separate regulation of exocytic insertion of two distinct transporters into the apical membrane.